Project Summary Exploring the biology of O-acetyl sialic acids using stable synthetic mimics This supplement request explores the possibility that sialoglycans may be co-receptors for SARS and COVID- 19 virus spike (S) proteins, as is the case with MERS and other Coronaviruses (CoVs). Numerous viruses recognize host cell surface glycans that terminate in sialic acids (Sias), a family of 9-carbon-backbone monosaccharides present at very high densities on all vertebrate cell surfaces, and on most secreted proteins? ?particularly mucins that line and protect mucosal surfaces like the airways. Viral recognition of host sialoglycans is affected by Sia type, linkage to, and the structure of underlying glycans. Much of this natural diversity of Sias in viral infection remains underexplored. While many respiratory disease-causing viruses target Sias, Sia recognition is not currently reported in SARS-CoV-2, the COVID-19 pandemic virus. This stands in contrast to the extensive literature on Coronaviruses and Sia receptors and is likely because a definitive human protein receptor (ACE2) for the virus S protein has been identified. A similar situation existed for the earlier MERS-CoV which had a well-defined receptor (DPP4) but was later found to also bind Sias via a different binding site. Given the very high Sia density in vivo, and the fact that Sias are the first contact of a virus on a mucosal surface, Sia diversity is likely to play important roles during natural infections. We hypothesize that airway Sias are also recognized by S proteins of SARS-CoV-1 and SARS-CoV-2. This supplement is based on strong foundations built by decades of studies of Sia diversity by the collaborating labs, including the parent project which addresses instability of Sia O-acetyl modifications by synthesizing sialosides with corresponding N-acetyl analogs. The urgent need for more careful exploration of Sia-binding functions of SARS and MERS will utilize a unique sialoglycan microarray built up over years of collaboration between the labs. Additional diversity of human sialosides such as those with 9-O-lactyl Sia that have heretofore not been studied, but could be critical, will also be explored. We propose sialoglycan microarray studies of recombinant soluble external domains of S proteins of MERS-CoV, SARS-CoV-1 and SARS-CoV-2 in comparison with human CoVs causing milder diseases, to detect Sia-dependent binding that has been missed so far. We will synthesize sialosides containing naturally occurring 9-O-lactyl-Sia and more stable 9-N-lactyl analogs and integrate these with the microarray and binding studies. Computational studies, including molecular dynamics simulation of binding free energies, will complement the array studies by predicting Sia variants that might bind, and modelling binding seen in array studies. These studies will generate new knowledge that may help to better understand viral infection, pathogenesis, and transmission from animals to humans and among humans. Additional viral epitopes critical for binding neutralization may be identified that could suggest novel preventative and/or therapeutic approaches to COVID-19. This project is therefore well suited to the urgency of the current pandemic situation. Information learned can also be applied toward the prediction and prevention of future epidemics and pandemics.